Benzo(e)pyrene is a cytotoxic chemical to the eyes, while neurohormone melatonin may exhibit protective effects on this cytotoxicity. In the current study, we have investigated the cytotoxic effects of benzo(e)pyrene on the chicken embryonic optic cups formation and whether melatonin supplementation protects chicken embryos against this xenobiotic toxicity. Fertilized chicken eggs were incubated for 48 h and then, they were divided into different groups. These groups included basal (without any treatment), control (distilled water), benzo(e)pyrene, melatonin and benzo(e)pyrene + melatonin groups, respectively. The 10 µl of distilled water or same volume of solution containing treatment compounds were injected into the air sac of the chicken egg. After an additional 18 h of incubation, the chicken embryos were excised and analyzed. The cytotoxicity was measured by a colorimetric whole chick embryo trypan blue assay. In embryos from basal, control and melatonin (0.01, 1 and 100 µM) groups, the frequency of the embryos with normal optic cups was 100% and had no increase in the embryonic cell death observed in post excision. In contrast, the frequency of normal optic cups in the benzo(e)pyrene (0.02 to 1200 µM) groups was significantly reduced (log IC50= -4.24 ± 0.02, R2= 0.98) with concentration-responsive manner. In addition, an increase in the embryonic cell death was also observed (log IC50 = -7.23 ± 0.28; R2 = 0.63). Melatonin treatment dose-responsively inhibited the benzo(e)pyrene-induced optic cups abnormality by 22.35 ± 4.06, 76.38 ± 3.30 and 100 % at the concentrations of 0.01, 1 and 100 µM, respectively. This same phenomenon was also observed in benzo(e)pyrene-induced embryonic cell death, i.e., melatonin suppressed the embryonic cell death by 16.67 ± 4.17, 54.17 ± 4.17 and 100 % with the abovementioned concentrations, respectively. Thus, melatonin supplementation injected into the chicken eggs protected against the benzo(e)pyrene embryotoxicity. Different pathways can be involved in melatonin’s protective effects.